Advanced Molecular Diagnostic Test for Neurofibromatosis ABSTRACT Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by mutations in the NF1 gene. In children, NF1 causes learning disabilities, coordination problems and benign tumors later in life that grow on nerve tissue. Currently more than 100,000 Americans suffer from NF1 with the disease having one of the highest prevalence rates for a genetic disorder (~1 out of 4,000 births). Progress has recently been made in identifying possible drugs for this disease including the proposed use of statins to alleviate learning disabilities in children with NF1. There is a critical need for a cost-effective test to screen for mutations in the NF1 gene. However, a significant problem is the wide variety of NF1 mutations which have been found to occur. Most inactivating mutations (>80%) are chain truncating and distributed throughout the gene. The preferred method to detect such mutations is the protein truncation test (PTT). However, because of the many limitations of conventional PTT, it is not practical for routine clinical use. Instead, current diagnosis is normally based on established non-genetic clinical criteria. Significant progress was made during Phase I in overcoming these limitations by developing a cost- effective alternative to conventional PTT. One approach utilizes an ELISA-based protein truncation test (ELISA-PTT) to detect chain-truncations. In contrast to conventional PTT, ELISA-PTT eliminates the need for electrophoresis and radioactivity. A second and complementary approach, based on mass spectrometric analysis of in vitro expressed proteins (MASSIVE-PRO), is able to additionally scan for missense mutations by precisely measuring the mass shift from single amino acid substitutions. Studies performed using these improved assays on normal and mutant cDNAs as well a limited number of NF1 patient samples revealed that these tests are highly effective with sensitivities comparable to and in some cases superior to standard DNA sequencing. During Phase II, we will further develop and extensively evaluate these novel NF1 diagnostic assays. The ELISA-PTT will be improved to allow scanning of the entire NF1 coding region using mRNA isolated from patient blood and a single-step PCR protocol. The MASSIVE-PRO assay will also be improved so that it can provide specific sequence information about mutations in the NF1 gene. Advanced technologies based on several innovations which can significantly reduce the number of PCR and cell-free translation reactions will also be evaluated. These assays, once optimized, will be extensively evaluated in collaboration with Dr. Ludwine Messiaen, Director of Genomics at the University of Alabama and a world leader in NF1 testing using a repository of validated genomic DNA and mRNA samples from NF1 patients. Clinical beta-testing will be performed during the third year in Dr. Messiaen's laboratory. In addition, Quest Diagnostics will work closely with AmberGen in order to assure these NF1 tests are properly designed for the requirements of the clinical laboratory environment. Advanced Molecular Diagnostic Test for Neurofibromatosis NARRATIVE Currently more than 100,000 Americans suffer from neurofibromatosis type 1 (NF1) which is among the most prevalent genetic disorders (~1 out of 4,000 births). An important goal is to develop a cost- effective test to screen for mutations in the NF1 gene. Significant progress was made during Phase I in developing a cost-effective alternative to conventional genetic tests. During Phase II, we propose to further develop and extensively evaluate these novel NF1 mutation assays in collaboration with Dr. Ludwine Messiaen, Director of Genomics at the University of Alabama and a world leader in NF1 testing using a repository of validated genomic DNA and mRNA samples. If successful, the test will enable more extensive screening and consequently increased surveillance and better management of NF1.